一种简便的小鼠精原干细胞分离培养方法

A Simple and Convenient Method of Mouse Spermatogonial Stem Cells Isolation and Culture

采用一步酶消化法分离小鼠精原干细胞,比较α-MEM、DMEM培养基对体外培养的精原干细胞生长状态的影响,对精原干细胞集落进行形态观察、碱性磷酸酶(alkaline phosphatase,AKP)染色和免疫组化鉴定,并诱导精原干细胞向精子细胞分化。结果显示,以小鼠胚胎成纤维细胞作为饲养层,用α-MEM培养的精原干细胞集落较大且呈葡萄串状或念珠状,细胞状态较好;小鼠精原干细胞集落的AKP染色阳性呈紫红色;在红色荧光下精原干细胞集落的Oct-4核蛋白表达为阳性、膜蛋白c-Kit、β_1-integrin和Gfrα-1表达为阳性;精原干细胞经维甲酸(all-trans-retinoic-acid,RA)诱导可初步分化成精子样细胞。因此,采用一步酶消化法能够分离小鼠精原干细胞,α-MEM更适合小鼠精原干细胞体外培养。

We isolated mouse spermatogonial stem cells （mSSCs） by the method of one-step enzymatic digestion, cultured mSSCs in vitro and compared the effect of α-MEM and DMEM on growth condition. The mSSCs colonies were identified by morphology observation, alkaline phosphatase （AKP） staining, immunohistochemistry and we induced the mSSCs differentiated into sperm-like cells. The results showed that using the mouse embryonic fibroblast cell as the feeder cell. the mSSCs colonies cultured by α-MEM were in good state and the graped-like or rosary-like; AKP staining showed the colour of mulberry as the positive; under the red fluorescent, the Oct-4 neucleoprotein of mSSCs colonies cells expressed positive and the membrane protein of C-Kit, α-integrin and Gfrα-1 expressed positive; the mSSCs treated with retinoic acid （RA） can be induced into the initial status of sperm-like cell. It was concluded that used the method of one-step enzymatic digestion could isolate mSSCs successfully and α-MEM was more suitable for mSSCs in vitro culture.